The fundamental function of a neuron is to receive, conduct, and transmit signals. Despite the varied purpose of the signals carried by different classes of neurons, the form of the signal is always the same and consists of changes in the electrical potential across the plasma membrane of the neuron. The plasma membrane of a neuron contains voltage-gated cation channels, which are responsible for generating this electrical potential (also referred to as an action potential or nerve impulse) across the plasma membrane.
One class of voltage-gated cation channels are the voltage-gated potassium channels (Kv). These include: (1) the delayed potassium channels, which repolarize the membrane after each action potential to prepare the cell to fire again; (2) the early potassium channels, which open when the membrane is depolarized and act to reduce the rate of firing at levels of stimulation which are just above the. threshold required for firing; and (3) the calcium-activated potassium channels, which act along with the voltage-gated calcium channels to decrease the response of the cell to an unchanging prolonged stimulation, a process called adaptation. In addition to being critical for action potential conduction, the voltage-gated potassium channels also play a role in neurotransmitter release. As a result of these activities, voltage-gated potassium channels are important in controlling neuronal excitability (Hille B., Ionic Channels of Excitable Membranes, Second Edition, Sunderland, Mass.: Sinauer, (1992)).
There is a surprising amount of structural and functional diversity within the voltage-gated potassium channels. This diversity is generated both by existence of multiple genes and by alternative splicing of RNA transcripts produced from the same gene. Nonetheless, the amino acid sequences of the known voltage-gated potassium channels show similarity. The Drosophila SH locus was the first potassium channel structural gene to be isolated (Kamb A. et al. (1987) Cell 50: 405). Since then, a number of additional potassium channel genes have been cloned from Drosophila and other organisms (Baumann A. et al. (1988) EMBO J. 7: 2457). One of these genes is the X-linked EAG locus, which was originally identified in Drosophil, on the basis of mutations that cause a leg-shaking phenotype (Kaplan W. D. et al. (1969) Genetics 61: 399). Electrophysiological studies revealed that EAG mutations caused spontaneous repetitive firing in motor axons and elevated transmitter release at the larval neuromuscular junction (Ganetzky B. et al. (1985) Trends Neurosci. 8:322). The striking hyperexcitability of EAG mutants demonstrates the importance of EAG channels in maintaining normal neuronal excitability in Drosophila (Ganetzky B. et al. (1983) J. Neurogeret. 1: 17-28).
EAG, along with m-EAG, ELK. and h-ERG define a family of potassium channel genes in Drosophila and mammals. A distinctive feature of the EAG/ERG family is the homology to cyclic nucleotide binding domains of cyclic nucleotide-gated cation channels and cyclic nucleotide-activated protein kinases (Kaupp, U. B. et al. (1991) Trends Neurosci. 14: 150-157). However, unlike the veirebrate cyclic nucleotide-gated cation channels, which are relatively voltage-insensitive, activation of EAG/ERG channels shows a very steep voltage dependence (Robertson, G. et al. (1993) Biophys. J. 64: 430). In addition, whereas cyclic nucleotide-activated cation channels show little selectivity among monovalent and divalent cations, eag is strongly selective for K+ over Na+. The EAG/ERG family may thus be an evolutionary link between voltage-activated potassium channels and cyclic nucleotide-gated cation channels with intermediate structural and functional properties.
The present invention is based, at least in part, on the discovery of novel ERG potassium channel family members, referred to herein as xe2x80x9cERG-like proteinsxe2x80x9d (xe2x80x9cERG-LPxe2x80x9d) nucleic acid and protein molecules. The ERG-LP molecules of the present invention are useful as targets for developing modulating agents to regulate a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding ERG-LP proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of ERG-LP-encoding nucleic acids.
In one embodiment, an ERG-LP nucleic acid molecule of the invention is at least 28%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:1, SEQ ID NO:3, or a complement thereof. In another embodiment, an ERG-LP nucleic acid molecule of the invention is at least 42%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:17, or a complement thereof. In another embodiment, an ERG-LP nucleic acid molecule of the invention is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more homologous. to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:7, SEQ ID NO:9, or a complement thereof.
In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:1 or 3, or a complement thereof. In another embodiment, the nucleic acid molecule: includes SEQ ID NO:3 and nucleotides 1-112 of SEQ ID NO:1. In another preferred embodiment, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:1 or 3. In another preferred embodiment, the nucleic acid molecule includes a fragment of at least 949 nucleotides of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or a complement thereof.
In another preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:4 or 6, or a complement thereof. In another embodiment, the nucleic acid molecule. includes SEQ ID NO:6 and nucleotides 1-214 of SEQ ID NO:4. In yet another embodiment, the nucleic acid molecule includes SEQ ID NO:6 and nucleotides 1844-2694 of SEQ ID NO:4. In another preferred embodiment, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:4 or 6. In another preferred embodiment, the nucleic acid molecule includes a fragment of at least 307 nucleotides of the nucleotide sequence of SEQ ID NO:4, SEQ ID NO:6, or a complement thereof.
In another preferred embodiment, the isolated nucleic acid molecule includes at least 200 consecutive. nucleotides, more preferably at least 400 consecutive nucleotides, more preferably at least 600 consecutive nucleotides, more preferably at least 800 consecutive nucleotides, more preferably at least 1000 consecutive nucleotides, more preferably at least 1200 consecutive nucleotides, more preferably at least 1400 consecutive nucleotides, more preferably at least 1500 consecutive nucleotides of the nucleotide sequence shown SEQ ID NO:4 or 6, or a complement thereof.
In another preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:7 or 9, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:9 and nucleotides 1-262 of SEQ ID NO:7. In another preferred embodiment, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:7 or 9. In another preferred embodiment, the nucleic acid molecule includes a fragment of at least 1114 nucleotides of the nucleotide sequence of SEQ ID NO:7, SEQ ID NO:9, or a complement thereof.
In another preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO: 15 or 17, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:17 and nucleotides 1-195 of SEQ ID NO:15. In yet another embodiment, the nucleic acid molecule includes SEQ ID NO:17 and nucleotides 3517-5107 of SEQ ID NO:15. In another preferred embodiment, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:15 or 17.
In another embodiment, an ERG-LP nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2. SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:16. In a preferred embodiment, an ERG-LP nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 37%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to the amino acid sequence of SEQ ID NO:2, or SEQ ID NO:8. In another preferred embodiment, an ERG-LP nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95% or more homologous to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:16.
In another preferred embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of human. or monkey ERG-LP1. In yet another preferred embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:2, or SEQ ID NO:8. In yet another preferred embodiment, the nucleic acid molecule, is at least 387 nucleotides in length and encodes a protein having an ERG-LP1 activity (,is described herein). In yet another preferred embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of human ERG-LP-2. In yet another preferred emnbodiment, the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:16.
Another embodiment of the invention features nucleic acid molecules, preferably ERG-LP nucleic acid molecules, which specifically detect ERG-LP nucleic acid molecules relative to nucleic acid molecules encoding non-ERG-LP proteins. For example, in one embodiment, such a nucleic acid molecule is at least 949, 950-1000, 1000-1050, 1050-1100 or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1, or a complement thereof. In preferred embodiments, the nucleic acid molecules are at least 15 (e.g., contiguous) nucleotides in length and hybridize under stringent conditions to nucleotides 1082-1100, 1258-1289, 1336-1343, 1404-1430, 2190-2428, or 3107-3355 of SEQ ID NO:1. In other preferred embodiments, the nucleic acid molecules comprise nucleotides 1082-1100, 1258-1289, 1336-1343, 1404-1330, 2190-2428, or 3107-3355 of SEQ ID NO:1.
In another particularly preferred embodiment, the nucleic acid molecule comprises a fragment of at least 307, 350-400, 400-450, 450-500 or more nucleotides of the nucleotide sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:17, or a complement thereof. In preferred embodiments, the nucleic acid molecules are at least 15 (e.g., contiguous) nucleotides in length and hybridize under stringent conditions to nucleotides 1-29, 442-621, 755-1013, 1170-1246, or 1463-1651 of SEQ ID NO:4. In other preferred embodiments, the nucleic acid molecules include nucleotides 1-29, 442-621, 755-1013, 1170-1246, or 1463-1651 of SEQ ID NO:4.
In other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:1 or SEQ ID NO:3 under stringent conditions. In yet other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:5, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:4 or SEQ ID NO:6 under stringent conditions. In other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:8, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:7 or SEQ ID NO:9 under stringent conditions. In yet other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:16, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:15 or SEQ ID NO:17 under stringent conditions.
Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to an ERG-LP nucleic acid molecule, e.g., the coding strand of an ERG-LP nucleic acid molecule.
Another aspect of the invention provides a vector comprising an ERG-LP nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector. In another embodiment, the invention provides a host cell containing a vector of the invention. The invention also provides a method for producing a protein, preferably an ERG-LP protein, by culturing in a suitable medium, a host cell, e.g., a mammals host cell such as a non-human mammalian cell, of the invention containing a recombinant expression vector, such that the protein is produced.
Another aspect of this invention features isolated or recombinant ERG-LP proteins and polypeptides. In one embodiment, the isolated protein, preferably an ERG-LP protein, includes at least one transmembrane domain. In another embodiment, the isolated protein, preferably an ERG-LP protein, includes a P-loop. In another embodiment, the isolated protein, preferably an ERG-LP protein, includes a cyclic nucleotide-binding domain. In another embodiment, the isolated protein, preferably an ERG-LP protein, includes transmembrane region cyclic nucleotide gated channel domain. In another embodiment, the isolated protein, preferably an ERG-LP protein, includes at least one transmembrane domain, a P-loop, a cyclic nucleotide-binding domain, and a transmembrane region cyclic nucleotide gated channel domain. In a preferred embodiment, the protein, preferably an ERG-LP protein, includes at least one transmembrane domain, a P-loop, and a cyclic nucleotide-binding domain, and has an amino acid sequence at least about 25%, 30%, 35%, 37%, 40%, 45%, 50%, 55%, 60%, 650%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, or more homologous to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:16. In another preferred embodiment, the protein, preferably an ERG-LP protein, includes at least one transmembrane domain and plays a role in generating an electrical potential across a plasma membrane, e.g., a neuronal plasma membrane or a muscle plasma membrane. In another preferred embodiment, the protein, preferably an ERG-LP protein, includes at least one P-loop and plays a role in generating an electrical potential across a plasma membrane, e.g., a neuronal plasma membrane or a muscle plasma membrane. In another preferred embodiment, the protein, preferably an ERG-LP protein, includes at least one cyclic nucleotide-binding domain, and plays a role in generating an electrical potential across a plasma membrane, e.g., a neuronal plasma membrane or a muscle plasma membrane. In another preferred embodiment, the protein, preferably an ERG-LP protein, includes at least one transmembrane region cyclic nucleotide gated channel domain and plays a role in generating an electrical potential across a plasma membrane, e.g., a neuronal plasma membrane or a muscle plasma membrane. In another preferred embodiment, the protein, preferably an ERG-LP protein, includes at least one transmembrane domain and a P-loop, and plays a role in generating an electrical potential across a plasma membrane, e.g., a neuronal plasma membrane or a muscle plasma membrane. In another preferred embodiment, the protein, preferably an ERG-LP protein, includes at least one transmembrane domain and a cyclic nucleotide-binding domain, and plays a role in generating an electrical. potential across a plasma membrane, e.g., a neuronal plasma membrane or a muscle plasma membrane. In another preferred embodiment, the protein, preferably an ERG-LP protein, includes at least one transmembrane domain and a transmembrane region cyclic nucleotide gated channel domain, and plays a role in generating an electrical potential across a plasma membrane, e.g., a neuronal plasma membrane or a muscle plasma membrane. In another preferred embodiment, the protein, preferably an ERG-LP protein, includes at least one P-loop and a cyclic nucleotide-binding domain, and plays a role in generating, an electrical potential across a plasma membrane, e.g., a neuronal plasma membrane or a muscle plasma membrane. In another preferred embodiment, the protein, preferably in ERG-LP protein, includes at least one P-loop and a transmembrane region cyclic nucleotide gated channel domain, and plays a role in generating an electrical potential across a plasma membrane, e.g., a neuronal plasma membrane or a muscle plasma membrane. In another preferred embodiment, the protein, preferably an ERG-LP protein, includes at least one transmembrane domain, a P-loop, and a cyclic nucleotide-binding domain, and plays a role in generating an electrical potential across a plasma membrane, e.g., a neuronal plasma membrane or a muscle plasma membrane. In another preferred embodiment, the protein, preferably an ERG-LP protein, includes at least one transmembrane domain, a P-loop, and a transmembrane region cyclic nucleotide gated channel domain, and l)lays a role in generating an electrical potential across a plasma membrane, e.g., a neuronal plasma membrane or a muscle plasma membrane. In yet another preferred embodiment, the protein, preferably an ERG-LP protein, includes at least one transmembrane domain, a P-loop, and a cyclic nucleotide-binding domain, and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:9.
In another preferred embodiment, the isolated protein includes at least 50 consecutive amino acids, more preferably at least 100 consecutive amino acids, more preferably at least 150 consecutive amino acids, more preferably at least 200 consecutive amino acids, more preferably at least 250 consecutive amino acids, more preferably at least 0.350 consecutive amino acids, more preferably at least 450 consecutive amino acids, more preferably at least 500 consecutive amino acids of the amino acid sequence shown SEQ ID NO:5 or 16.
In another embodiment, the invention features fragments of the proteins having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8 or SEQ ID NO:16, wherein the fragment comprises at least 15 amino acids (e.g., contiguous amino acids) of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO:16. In another embodiment, the protein, preferably an ERG-LP protein, has the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8 or SEQ ID NO:16.
In another embodiment, the invention features an isolated protein, preferably an ERG-LP protein, which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 28%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, 95% or more homologous to a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or a complement thereof. In yet another embodiment, the invention features an isolated protein, preferably an ERG-LP protein, which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 42%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to a nucleotide sequence of SEQ ID NO:4, SEQ ID NO:6, or a complement thereof. In yet another embodiment, the invention features an isolated protein, preferably an ERG-LP protein, which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to a nucleotide sequence of SEQ ID NO:7, SEQ ID NO:9, or a complement thereof. In yet another embodiment, the invention features an isolated protein, preferably an ERG-LP protein, which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homologous to a nucleotide sequence of SEQ ID NO:15, SEQ ID NO:17, or a complement thereof. This invention further features an isolated protein, preferably an ERG-LP protein, which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:17 or a complement thereof.
The proteins of the present invention or biologically active portions thereof, can be operatively linked to a non-ERG-LP polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins. The invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind proteins of the invention, preferably ERG-LP proteins. In addition, the ERG-LP proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
In another aspect, the present invention provides a method for detecting the presence of an ERG-LP nucleic acid molecule, protein or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting an ERG-LP nucleic acid molecule, protein or polypeptide such that the presence of an ERG-LP nucleic acid molecule, protein or polypeptide is detected in the biological sample.
In another aspect, the present invention provides a method for detecting the presence of ERG-LP activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of EPG-LP activity such that the presence of ERG-LP activity is detected in the biological sample.
In another aspect, the invention provides a method for modulating ERG-LP activity comprising contacting a cell capable o)f expressing ERG-LP with an agent that modulates ERG-LP activity such that ERG-LP activity in the cell is modulated. In one embodiment, the agent inhibits ERG-LP activity. In another embodiment, the agent stimulates ERG-LP activity. In one embodiment, the agent is an antibody that specifically binds to an ERG-LP protein. In another embodiment, the agent modulates expression of ERG-LP by modulating transcription of an ERG-LP gene or translation of an ERG-LP mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of an ERG-LP mRNA or an ERG-LP gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant ERG-LP protein or nucleic acid expression or activity by administering an agent which is an ERG-LP modulator to the subject. In one embodiment, the ERG-LP modulator is an ERG-LP protein. In another embodiment the ERG-LP modulator is an ERG-LP nucleic acid molecule. In yet another embodiment, the ERG-LP modulator is a peptide, peplidomimetic, or other small molecule. In a preferred embodiment, the disorder characterized by aberrant ERG-LP protein or nucleic acid expression is a CNS disorder.
The present invention also provides a diagnostic assay for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding an ERG-LP protein; (ii) mis-regulation of the gene; and (iii) aberrant post-translational modification of an ERG-LP protein, wherein a wild-type form of the gene encodes an protein with an ERG-LP activity.
In another aspect the invention provides a method for identifying a compound that binds to or modulates the activity of an ERG-LP protein, by providing an indicator composition comprising an ERG-LP protein having ERG-LP activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on ERG-LP activity in the indicator composition to identify a compound that modulates the activity of an ERG-LP protein.
Other features and advantages of the invention will be apparent from the following detailed description and claims.